It is well known that carbohydrate structures of various complexities are the antigenic determinants for a wide range of substances. It is also well established that relatively small molecules, known as haptens, can correspond to the structure of the antigenic determinant. The hapten, when attached to an appropriate carrier molecule, provides an artificial antigen which, when administered to an animal under appropriate conditions, will give rise to the production of antibodies having a specificity for the hapten. Furthermore, in recent years, much art has developed for the preparation of immunoabsorbents from haptens. This art involves the attachment of the hapten, normally through covalent bonding but at times through hydrophobic bonding, to a solid, latex or gelatinous support. Thus, the hapten is immobilized so that when the resulting immunoabsorbent is exposed to antibodies with combining sites for the haptenic structure, the antibodies will attach themselves to the surface of the immunoabsorbent and thereby be specifically removed from solution.
Many varieties of solid, latex and gel supports for the preparation of immunoabsorbents have been developed and many ways have been devised for attachment of the hapten to these insoluble structures. Although improvements in these matters are possible, the main problem remains of having simple access to the desired hapten in a form convenient for attachment to the carrier molecule.
It was the original purpose of our work to develop a practical process for the synthesis of D-galactosamine hydrochloride (XXXVII) and of D-lactosamine hydrochloride (XXXIX) and derivatives of these. Both galactosamine and lactosamine, usually in the form of their N-acetylated derivatives, are found widespread in nature. They occur in glycoproteins, glycolipids and mucopolysaccharides. As such they are important building units found in the blood group substance antigenic determinants.
The main prior art source of D-galactosamine is the acid hydrolysis of chrondroitin sulfate C which is obtained by extracting cartilaginous tissues such as tendons, trachea and nasal septa. These yields are uncertain and it is difficult to obtain a crystalline product. Numerous chemical syntheses exist which include the opening of 1,6:2,3-dianhydro-.beta.-D-talopyranose with ammonia or with azide ion. However, these methods involve six to eleven separate chemical transformations starting from the simple sugars. Shorter methods depend upon rather rare sugars as starting materials.
Inversion of the C-4 configuration of glucosamine through displacement of a 4-O-sulfonate of 2-acetamido-2-deoxy glucopyranosyl derivatives has also been utilized for the synthesis of D-galactosamine. However, the elaboration of glucosamine to the necessary starting material is tedious.
The synthesis of lactosamine is more difficult as it necessarily involves a glycosylation of a galactosyl halide with an elaborate derivative of 2-acetamido-2-deoxy-glucose. The most recently published method requires nine chemical transformations, starting from 2-acetamido-2-deoxy glucosamine, prior to the glycosylation step.
In accordance with a feature of the present invention, there is provided a reagent that allows efficient and high yield preparations of glycosides which contain the 2-acetamido-2-deoxy-.alpha.-D-galactopyranosyl group which is found, for example, in the antigenic determinant for the human A blood group and the Forssman antigen. The reagent thus claimed useful is 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.beta.-D-galactopyranosyl chloride (XXIII) prepared simply from D-galactal triacetate (I) in high yield.
It has long been anticipated that the use of a .beta.-glycosyl halide would tend to yield the .alpha.-(1,2-cis)-glycosidic linkage through Walden inversion of the reacting center under Koenings-Knorr reaction conditions when the 2-substituent is so chosen as to not participate in a reaction at the anomeric center. Thus, for example, Wolfrom, Thompson and Linebeck (J. Org. Chem., 28, 860 (1963)) developed tri-O-acetyl-2-nitro-.beta.-D-glucopyranosyl chloride for the purpose of synthesizing .alpha.-D-glucopyranosides. Indeed, several papers have appeared in the recent literature which utilize 2-azido-2-deoxy-.beta.-D-glycopyranosyl chlorides such as is reported in processes of this invention leading to the formation of 2-azido-2-deoxy-.alpha.-D-galactopyranosides. However, it must be noted that the processes reported by Paulsen and co-workers (Angew. Chem., Int. Ed., 14, 558 (1975); Tet. Lett., 1493 (1975) and 2301 (1976); Angew. Chem., Int. Ed., 15, 440 (1975)) are of limited, if any commercial value in view of the extreme difficulty in achieving the synthesis of the desired 2-azido-2-deoxy reagent; namely, 6-O-acetyl-2-azido-3,4-O-benzyl-2-deoxy-.beta.-D-galactopyranosyl chloride.
This invention reports a novel process for preparing efficiently the compound 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.beta.-D-galactopyranosyl chloride (XXIII) and its engagement in reactions with alcohol to form 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.alpha.-D-galactopyranosides (A) under appropriate Koenings-Knorr type conditions for the condensation. The invention in ##STR1## part concerns the discovery of processes that render compound XXIII a readily available reagent for use in reactions leading to products of type A. Thus, it has become commercially feasible to synthesize the terminal trisaccharide antigenic determinant for the human A-blood as is present in structures B for the type 1 and type 2 antigenic determinants for the human A blood group. The trisaccharide is synthesized in a form useful for the preparation of artificial antigens and immunobsorbents related to the human A blood group. ##STR2##
The formation of .alpha.-azido-.beta.-nitratoalkanes from the reaction of olefins with sodium azide and ceric ammonium nitrate has been reported by Trahanovsky and Robbins (J.Am. Chem. Soc., 93, 5256 (1971)). However the extension of the above reaction to vinylic ethers or structures as complex as D-galactal triacetate is not obvious. The base of this invention was the discovery that the addition of the azide and nitrate groups to 1,2-unsaturated sugars can be made to proceed in high economical yield to form the 2-azido-2-deoxy glycosyl nitrate.